The present invention features compositions, reagents and methods for enhancing an amplification protocol. Preferably, the compositions, reagents and methods are used in conjunction with an RNA polymerase driven transcription-associated amplification protocol.
None of the references described herein are admitted to be prior art to the claimed invention.
Nucleic acid amplification involves the enzymatic synthesis of nucleic acid amplicons that contain a sequence complementary to a nucleic acid sequence being amplified. Nucleic acid amplification can be performed using different techniques such as those involving transcription-associated amplification, the polymerase chain reaction (PCR), ligase chain reaction (LCR) and strand displacement amplification (SDA).
Uses of nucleic acid amplification include diagnostic and synthetic applications. Diagnostic applications of nucleic acid amplification typically involve screening for whether amplicons are produced, the amount of amplicon produced, and/or determining whether produced amplicons contain a particular sequence.
Transcription-associated amplification of a nucleic acid sequence generally employs an RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and a promoter-template complementary oligonucleotide. The promoter-template complementary oligonucleotide contains a 5xe2x80x2 sequence recognized by an RNA polymerase and a 3xe2x80x2 sequence that hybridizes to a template nucleic acid in a location 3xe2x80x2 of a target sequence that is sought to be amplified. After hybridization of the promoter-template complementary oligonucleotide to the template, a double-stranded promoter is formed upstream from the target sequence. Double-stranded promoter formation generally involves DNA polymerase activity.
RNA polymerase-associated amplification is initiated by the binding of an RNA polymerase to a promoter region that is usually double-stranded. The RNA polymerase proceeds downstream from the promoter region and synthesizes ribonucleic acid in a 5xe2x80x2 to 3xe2x80x2 direction. Multiple copies, generally in the range of 100-3,000 RNA transcripts, can be produced by RNA polymerase-associated amplification using a single template.
Different formats can be employed for performing transcription-associated amplification. Examples of different formats are provided in publications such as Burg et al., U.S. Pat. No. 5,437,990; Kacian et al., U.S. Pat. No. 5,399,491; Kacian et al., U.S. Pat. No. 5,554,516; Kacian et al., International Application No. PCT/US93/04015, International Publication No. WO 93/22461; Gingeras et al., International Application No. PCT/US87/01966, International Publication No. WO 88/01302; Gingeras et al., International Application No. PCT/US88/02108, International Publication No. WO 88/10315; Davey and Malek, European Application No. 88113948.9, European Publication No. 0 329 822 A2; Malek et al., U.S. Pat. No. 5,130,238; Urdea, International Application No. PCT/US91/00213, International Publication No. WO 91/10746; McDonough et al., International Application No. PCT/US93/07138, International Publication No. WO 94/03472; and Ryder et al., International Application No. PCT/US94/08307, International Publication No. WO 95/03430. (Each of these references is hereby incorporated by reference herein.)
PCR amplification is described by Mullis et al., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159, and in Methods in Enzymology, 155:335-350 (1987). (Each of these references is hereby incorporated by reference herein.)
An example of LCR is described in European Patent Publication No. 320 308 (hereby incorporated by reference herein). LCR uses at least four separate oligonucleotides. Two of the oligonucleotides hybridize to a nucleic acid template so that the 3xe2x80x2 end of one oligonucleotide and the 5xe2x80x2 end of the other oligonucleotide are positioned for ligation. The hybridized oligonucleotides are then ligated forming a full-length complement to the target sequence in the nucleic acid template. The double-stranded nucleic acid is then denatured, and third and fourth oligonucleotides are hybridized to the complementary strand and joined together. Amplification is achieved by further cycles of hybridization, ligation, and denaturation, producing multiple copies of the target sequence and the sequence complementary to the target sequence.
SDA is an isothermal amplification reaction based on the ability of a restriction enzyme to nick the unmodified strand of a hemiphosphorothioate form of its recognition site, and on the ability of a DNA polymerase to initiate replication at the nick and displace a downstream non-template strand. (See, e.g., Walker, PCR Methods and Applications, 3:25-30 (1993), Walker et al., Nucleic Acids Res., 20:1691-1996 (1992), and Walker et al., Proc. Natl. Acad. Sci., 89:392-396 (1991). Each of these references is hereby incorporated by reference herein.) The steps used in generating fragments for carrying out autocatalytic SDA amplification are indicated to be adaptable for generating fragments for transcription-associated amplification or amplification carried out using Q-beta technology. (Walker et al., Nucleic Acids Res., 20:1691-1696 (1992).)
The present invention features inhibitors of target-independent amplification and the use of such inhibitors for enhancing an amplification protocol. The inhibitors are believed to enhance an amplification protocol by inhibiting the ability of one or more nucleic acid polymerases to use nucleic acid in a polymerase reaction in the absence of target nucleic acid.
xe2x80x9cTarget-independent amplificationxe2x80x9d refers to the amplification of a nucleic acid sequence that is not a target nucleic acid sequence. The target nucleic acid sequence is present on a target nucleic acid and is a nucleotide base sequence, or region, sought to be amplified.
It is believed that the present invention benefits nucleic acid amplification by using competitors of amplification oligonucleotides to sequester amplification enzymes in solution from non-target nucleic acid such as amplification oligonucleotides. The competitors appear to compete with amplification oligonucleotides for binding to one or more amplification enzymes, and may be added in an excess amount relative to the amplification oligonucleotides.
In the absence of target nucleic acid, affected amplification enzymes are occupied by the competitors, and the ability of the enzymes to participate in a polymerase reaction involving non-target nucleic acid is inhibited. Amplification oligonucleotides hybridized to target nucleic acid favorably compete with the competitors for amplification enzyme binding. Thus, the competitors function as reversible inhibitors of amplification enzymes.
Amplification enzymes are nucleic acid polymerases that catalyze the synthesis of polynucleotides by polymerizing nucleoside triphosphates. Reversible inhibition of amplification enzymes is carried out to prevent the formation of undesirable side-products, such as one or more of the following: (1) a primer-dimer; (2) an RNA replicating nucleic acid; (3) a single-stranded primer extended RNA or DNA; and (4) a modification to a primer rendering the primer unable to participate in the amplification of a target nucleic acid sequence. The types of undesirable side-products that can be formed will depend upon the particular amplification protocol that is performed.
Amplification oligonucleotides hybridize to target nucleic acid and participate in an amplification reaction. Examples of amplification oligonucleotides include template-complementary probes, such as primers, and promoter-template complementary probes, such as promoter-primers.
While inhibitors of target-independent amplification described by the present invention are expected to function by competing with amplification oligonucleotides for binding to an amplification enzyme, unless otherwise specified in the claims, the claims are not limited to a particular mechanism. For example, probes having a high degree of sequence similarity to an RNA polymerase promoter which enhance an amplification protocol are described in the examples provided below. Such examples illustrate the effectiveness of such probes and allow for probe design based on sequence similarity to an RNA polymerase promoter without determining enzyme binding or the ability to compete with amplification oligonucleotides.
Thus, a first aspect of the present invention describes a decoy probe comprising,
a first nucleotide base recognition sequence region, wherein the first region binds to an RNA polymerase, and
an optionally present second nucleotide base recognition sequence region,
provided that if the first region is nucleic acid, then the second region is either directly joined to the 5xe2x80x2 end of the first region or is joined to the 3xe2x80x2 end or 5xe2x80x2 end of the first region by a non-nucleotide linker,
wherein the optionally present second region is present if the first region can be used to produce a functional double-stranded promoter sequence using a complementary oligonucleotide,
further provided that if the first region is nucleic acid which can be used to produce the functional double-stranded promoter sequence using the complementary oligonucleotide, then the decoy probe does not have a nucleic acid sequence greater than about 10 nucleotides in length joined directly to the 3xe2x80x2 end of the first region.
The first region contains a nucleotide base recognition sequence region to which an RNA polymerase can bind. An example of such a sequence is one sense of a double-stranded promoter sequence. The first region can contain, for example, a derivative of one sense of a promoter region, where the derivative cannot be used to produce a functional double-stranded promoter sequence when made double-stranded.
If the decoy probe can form a functional promoter, then it is desirable not to have significant downstream sequences that can be used as an amplifiable template. Preferably, if the first region can be used to produce a functional double-stranded promoter then the decoy probe does not have a nucleotide base sequence greater than 5 nucleotides in length joined directly to the 3xe2x80x2 end of the first region.
The presence of a second region positioned 3xe2x80x2 or 5xe2x80x2 to the first region does not prevent other regions from being present. For example, the decoy probe may contain a second region 3xe2x80x2 to the first region and may also contain a region joined either directly, or through a non-nucleotide linker, to the 5xe2x80x2 end of the first region.
Preferably, the decoy probe is a purified probe. By xe2x80x9cpurifiedxe2x80x9d is meant that the decoy probe makes up at least 0.1% of the recognition molecules present in a preparation. In preferred embodiments, the decoy probe makes up at least 1%, at least 5%, at least 25%, at least 50%, at least 75%, or 100% of the nucleic acid present in a preparation.
A xe2x80x9cnucleotide base sequence recognition moleculexe2x80x9d is a molecule containing nucleotide base recognition groups linked together by a backbone. Examples of nucleotide base sequence recognition molecules include peptide nucleic acids, oligonucleotides, and derivatives thereof. A nucleotide base recognition group can hydrogen bond to adenine, guanine, cytosine, thymine or uracil. The backbone presents the nucleotide base recognition groups in a proper conformation for hydrogen bonding to a complementary nucleotide present in a nucleic acid sequence.
A xe2x80x9cfunctional double-stranded promoter sequencexe2x80x9d is a sequence that is recognized by an RNA polymerase and can be used to produce readily detectable RNA transcripts. A functional double-stranded promoter can be formed from a single-stranded promoter sequence, for example, by hybridizing to the promoter sequence a complementary oligonucleotide.
A xe2x80x9cnon-nucleotide linkerxe2x80x9d refers to one or more chemical moieties which form a stable linkage under amplification conditions and which do not contain a nucleotide base recognition group that can act as a template in a polymerase reaction.
Another aspect of the present invention describes a decoy probe comprising,
a first nucleotide base recognition sequence region, wherein the first region has at least 35% sequence similarity to an RNA polymerase promoter sequence, and
an optionally present second nucleotide base recognition sequence region,
provided that if the first region is nucleic acid, then the second region is either directly joined to the 5xe2x80x2 end of the first region or is joined to the 3xe2x80x2 end or 5xe2x80x2 end of the first region by a non-nucleotide linker,
wherein the optionally present second region is present if the first region can be used to produce a functional double-stranded promoter sequence using a complementary oligonucleotide,
further provided that if the first region is nucleic acid which can be used to produce the functional double-stranded promoter sequence using the complementary oligonucleotide, then the decoy probe does not have a nucleic acid sequence greater than about 10 nucleotides in length joined directly to the 3xe2x80x2 end of the first region.
Decoy probe binding to an RNA polymerase can be measured using standard techniques, such as through the use of competitive and noncompetitive assays employing a labeled oligonucleotide having an RNA polymerase promoter sequence. Additionally, oligonucleotides binding to RNA polymerase can be selected for and produced in large quantities using the xe2x80x9cProtein Binding Amplification Protocolxe2x80x9d described infra.
Another aspect of the present invention describes a reagent mixture for use in an amplification reaction. The mixture contains a nucleic acid polymerase and a reversible inhibitor of the polymerase. The mixture does not contain a nucleic acid substantially complementary to the inhibitor. Thus, the mixture does not contain a nucleic acid that would hybridize to the inhibitor under the amplification conditions in which the mixture is employed.
The reagent mixture is particularly useful for providing an opportunity for the reversible inhibitor to bind with the amplification nucleic acid polymerase prior to exposure to amplification oligonucleotides. Preferably, the mixture does not contain the target sequence to be amplified.
Another aspect of the present invention describes an amplification procedure for amplifying a target nucleic acid sequence comprising the steps of:
a) producing a mixture comprising an amplification enzyme and a reversible inhibitor of the enzyme, where the reversible inhibitor does not hybridize to a target nucleic acid comprising the target nucleic acid sequence under amplification conditions and the mixture does not contain the target nucleic acid,
b) providing the mixture to the target nucleic acid, and
c) amplifying the target nucleic acid sequence under the amplification conditions.
Another aspect of the present invention describes a transcription-associated amplification procedure comprising the step of amplifying a nucleic acid sequence to produce multiple copies of RNA transcripts by combining together, under transcription-associated amplification conditions, a mixture comprising a target nucleic acid comprising the target nucleic acid sequence, a promoter-template complementary probe, a DNA polymerase, an RNA polymerase, ribonucleoside triphosphates, deoxyribonucleoside triphosphates, and means for reversibly inhibiting the RNA polymerase. The means for reversibly inhibiting the RNA polymerase does not hybridize to the target nucleic acid under the amplification conditions to form a stable inhibitor:target complex.
xe2x80x9cMeans for reversibly inhibitingxe2x80x9d refers to material described in the present application and equivalents thereof that can reversibly inhibit the activity of an amplification enzyme.
Another aspect of the present invention describes an improved method of amplifying a target nucleic acid sequence. The improvement comprises the step of providing a nucleic acid polymerase used in the amplification with means for reversibly inhibiting the polymerase prior to providing the polymerase to a target nucleic acid comprising the target nucleic acid sequence.
Expected advantages of the present invention include one or more of the following:
(1) increased yield of target complementary amplicons; (2) increased sensitivity; and (3) increased availability of polymerases for target amplification. Such advantages are expected to arise from the reduction of undesirable side-products.
Another advantage of the present invention is that it can be employed at an essentially constant temperature. At an essentially constant temperature, a reaction is not cycled between a high and a low temperature to alternatively denature and anneal nucleic acid, such as that occurring in PCR.
Various examples are used throughout the application. These examples are not intended in any way to limit the claimed invention.
Other features and advantages of the invention will be apparent from the following figures, detailed description of the invention, examples, and the claims.